CD4 T cells play a key role in the induction of immune responses, recognizing peptides derived from foreign antigens complexed to MHC class II on the surface of APCs. Most of the MHC molecules expressed on the cell surface are occupied by peptides derived from endogenous proteins. In order to be recognized by T cells, foreign peptides need to compete with self peptides for binding to MHC molecules. Self peptide:MHC complexes are involved in negative and positive selection of T cell receptor repertoire. In T cell-mediated autoimmunity, self peptide:MHC complexes become targets of autoreactive T cells. The overall goal of this project is to study generation of self peptide:MHC complexes and their influence on the T cell repertoire. Four specific questions will be asked. First, what are the characteristics of binding of naturally processed peptides to MHC class II molecule in terms of peptide and MHC class II structure, kinetics, and pH- dependence? Second, how is a self peptide:MHC class II complex generated in living cells? Third, how is tolerance to self peptide:MHC class II complexes established? Finally, what is the repertoire of self peptides bound to MHC class II expressed by different tissues in vitro and in vivo, and how do allelic differences in MHC class II influence the self peptide repertoire? To carry out these studies, we recently developed a technique of identification of self peptides associated with MHC class II and a monoclonal antibody specific for one such peptide:MHC complex. A combination of biochemical and molecular methods with quantitative immunochemical and FACS analysis of peptide:MHC class II interaction will be used to answer the above questions. Existing transgenic mice with tissue-specific expression of the peptide donor protein will be used to study T cell tolerance to the peptide. The influence of the level of expression on tolerance to the peptide:MHC class II complex will be studied by chronic in vivo injection of monoclonal antibody specific to this complex. In order to generalize these studies, other abundant peptides bound to MHC class II on various tissues and haplotypes will be identified, and monoclonal antibodies to other peptide:MHC class II complexes will be generated.